Inhibition of early RNA replication in Chikungunya and Dengue virus by lycorine: In vitro and in silico studies.

Arboviruses such as chikungunya virus (CHIKV) and dengue virus (DENV) collectively afflict millions of individuals worldwide particularly in endemic countries like India, leading to substantial morbidity and mortality. With the lack of effective vaccines for both CHIKV and DENV in India, the search for antiviral compounds becomes paramount to control these viral infections. In line with this, our investigation was focused on screening natural compounds for their potential antiviral activity against CHIKV and DENV. Using different assays, including plaque assay, immunofluorescence, and reverse transcription-quantitative real-time PCR (qRT-PCR), out of 109 natural compounds tested, we confirmed lycorine's in vitro antiviral activity against CHIKV and DENV at low micromolar concentrations in different cell types. Time of addition assays indicated that lycorine does not impede viral entry. Additionally, qRT-PCR results along with time of addition assay suggested that lycorine interferes with the synthesis of negative strand viral RNA. Molecular docking analysis was done to understand the mode of inhibition of viral replication. The results revealed that the most likely binding site with the highest binding affinity of lycorine, was at the palm and finger domains, in the vicinity of the catalytic site of CHIKV and DENV RNA-dependent RNA polymerase (RdRp). Collectively, our data underscores the potential of lycorine to be developed as a direct acting inhibitor for DENV and CHIKV, addressing the critical need of requirement of an antiviral in regions where these viruses pose significant public health threats.

A broadly neutralizing monoclonal antibody overcomes the mutational landscape of emerging SARS-CoV-2 variants of concern.

The emergence of new variants of SARS-CoV-2 necessitates unremitting efforts to discover novel therapeutic monoclonal antibodies (mAbs). Here, we report an extremely potent mAb named P4A2 that can neutralize all the circulating variants of concern (VOCs) with high efficiency, including the highly transmissible Omicron. The crystal structure of the P4A2 Fab:RBD complex revealed that the residues of the RBD that interact with P4A2 are a part of the ACE2-receptor-binding motif and are not mutated in any of the VOCs. The pan coronavirus pseudotyped neutralization assay confirmed that the P4A2 mAb is specific for SARS-CoV-2 and its VOCs. Passive administration of P4A2 to K18-hACE2 transgenic mice conferred protection, both prophylactically and therapeutically, against challenge with VOCs. Overall, our data shows that, the P4A2 mAb has immense therapeutic potential to neutralize the current circulating VOCs. Due to the overlap between the P4A2 epitope and ACE2 binding site on spike-RBD, P4A2 may also be highly effective against a number of future variants.

Analysis of domain organization and functional signatures of trypanosomatid keIF4Gs.

Translation initiation is the first step in three essential processes leading to protein synthesis. It is carried out by proteins called translation initiation factors and ribosomes on the mRNA. One of the critical translation initiation factors in eukaryotes is eIF4G which is a scaffold protein that helps assemble translation initiation complexes that carry out translation initiation which ultimately leads to polypeptide synthesis. Trypanosomatids are a large family of kinetoplastids, some of which are protozoan parasites that cause diseases in humans through transmission by vectors. While the protein translation mechanisms in eukaryotes and prokaryotes are well understood, the protein translation factors and mechanisms in trypanosomatids are poorly understood necessitating further studies. Unlike other eukaryotes, trypanosomatids contain five eIF4G orthologues with diversity in length and sequences. Here, I have used bioinformatics tools to look at trypanosomatid keIF4G orthologue sequences and report that there are similarities and considerable differences in their domains/motifs organization and signature amino acid sequences that are required for different functions as compared to human eIF4G. My analysis suggests that there is likely to be considerable diversity and complexity in trypanosomatid keIF4G functions as compared to other eukaryotes.

Identification of an anti-SARS-CoV-2 receptor-binding domain-directed human monoclonal antibody from a naïve semisynthetic library.

There is a desperate need for safe and effective vaccines, therapies, and diagnostics for SARS- coronavirus 2 (CoV-2), the development of which will be aided by the discovery of potent and selective antibodies against relevant viral epitopes. Human phage display technology has revolutionized the process of identifying and optimizing antibodies, providing facile entry points for further applications. Herein, we use this technology to search for antibodies targeting the receptor-binding domain (RBD) of CoV-2. Specifically, we screened a naïve human semisynthetic phage library against RBD, leading to the identification of a high-affinity single-chain fragment variable region (scFv). The scFv was further engineered into two other antibody formats (scFv-Fc and IgG1). All three antibody formats showed high binding specificity to CoV-2 RBD and the spike antigens in different assay systems. Flow cytometry analysis demonstrated specific binding of the IgG1 format to cells expressing membrane-bound CoV-2 spike protein. Docking studies revealed that the scFv recognizes an epitope that partially overlaps with angiotensin-converting enzyme 2 (ACE2)-interacting sites on the CoV-2 RBD. Given its high specificity and affinity, we anticipate that these anti-CoV-2 antibodies will be useful as valuable reagents for accessing the antigenicity of vaccine candidates, as well as developing antibody-based therapeutics and diagnostics for CoV-2.

Tetramerizing tGCN4 domain facilitates production of Influenza A H1N1 M2e higher order soluble oligomers that show enhanced immunogenicity in vivo.

One strategy for the development of a next generation influenza vaccine centers upon using conserved domains of the virus to induce broader and long-lasting immune responses. The production of artificial proteins by mimicking native-like structures has shown to be a promising approach for vaccine design against diverse enveloped viruses. The amino terminus of influenza A virus matrix 2 ectodomain (M2e) is highly conserved among influenza subtypes, and previous studies have shown M2e-based vaccines are strongly immunogenic, making it an attractive target for further exploration. We hypothesized that stabilizing M2e protein in the mammalian system might influence the immunogenicity of M2e with the added advantage to robustly produce the large scale of proteins with native-like fold and hence can act as an efficient vaccine candidate. In this study, we created an engineered construct in which the amino terminus of M2e is linked to the tetramerizing domain tGCN4, expressed the construct in a mammalian system, and tested for immunogenicity in BALB/c mice. We have also constructed a stand-alone M2e construct (without tGCN4) and compared the protein expressed in mammalian cells and in Escherichia coli using in vitro and in vivo methods. The mammalian-expressed protein was found to be more stable, more antigenic than the E. coli protein, and form higher-order oligomers. In an intramuscular protein priming and boosting regimen in mice, these proteins induced high titers of antibodies and elicited a mixed Th1/Th2 response. These results highlight the mammalian-expressed M2e soluble proteins as a promising vaccine development platform.

Taking a re-look at cap-binding signatures of the mRNA cap-binding protein eIF4E orthologues in trypanosomatids.

Protein translation leading to polypeptide synthesis involves three distinct events, namely, initiation, elongation, and termination. Translation initiation is a multi-step process that is carried out by ribosomes on the mRNA with the assistance of a large number of proteins called translation initiation factors. Trypanosomatids are kinetoplastidas (flagellated protozoans), some of which cause acute disease syndromes in humans. Vector-borne transmission of protozoan parasites like Leishmania and Trypanosoma causes diseases that affect a large section of the world population and lead to significant morbidity and mortality. The mechanisms of translation initiation in higher eukaryotes are relatively well understood. However, structural and functional conservation of initiation factors in trypanosomatids are only beginning to be understood. Studies carried out so far suggests that at least in Leishmania and Trypanosoma eIF4E function may not be restricted to canonical translation initiation and some of the homologues may have alternate/non-canonical functions. Nonetheless, all of them bind the cap analogs, albeit with different efficiencies, indicating that this property may play an important role in the functionality of eIF4Es. Here, I give a brief background of trypanosomatid eIF4Es and revisit the cap-binding signatures of eIF4E orthologues in trypanosomatids, whose genome sequences are available, in detail, in comparison to human eIF4E1 and Trypanosoma cruzi eIF4E5, with an expanded list of members of this group in light of newer findings. The group 1 and 2 eIF4Es may use either a variation of heIF4E1 or T. cruzi eIF4E5 cap-4-binding signatures, while eIF4E5 and eIF4E6 use distinct amino acid contacts.

Cell surface ectodomain integrity of a subset of functional HIV-1 envelopes is dependent on a conserved hydrophilic domain containing region in their C-terminal tail.

BACKGROUND: HIV-1 Env gp160 is cleaved to form gp120 and gp41 and the functional HIV-1 Env is a trimer of non-covalently associated heterodimeric subunits, gp120 and gp41. The cleaved, native, trimeric form of Envs expose only broadly neutralizing antibody (bNAb) epitopes while occluding epitopes targeted by non-neutralizing antibodies (non-NAbs). We and others have previously observed that efficient cleavage of Envs into their constituent subunits co-relates with specific binding to bNAbs and poor binding to non-neutralizing antibodies (non-NAbs). Such Envs have been identified from clades A, B and C which make up a majority of globally circulating HIV-1 strains. Frequently, the C-terminal tail (CT) of Envs is deleted to enhance expression and stabilize soluble Env-based vaccine immunogens. Deletion of CT of efficiently cleaved Indian clade C Env 4-2.J41 results in recognition by both NAbs and non-NAbs. It is to be noted that uncleaved Envs bind to both NAbs and non-NAbs. So we investigated whether altered antigenicity upon CT deletion of efficiently cleaved Envs is due to inefficient cleavage or conformational change as the mechanism by which the CT regulates the ectodomain (ET) integrity is not well understood. RESULTS: We studied the effect of CT deletion in four membrane bound efficiently cleaved Envs, A5 (clade A), 4-2.J41 (clade C), JRFL and JRCSF (clade B). Deletion of CT of the Envs, JRCSF and 4-2.J41, but not JRFL and A5 alter their ET antigenicity/conformation without affecting the cleavage efficiency. We carried out a series of deletion mutation in order to determine the region of the CT required for restoring native-like antigenicity/conformation of the ET of 4-2.J41 and JRCSF. Extending the CT up to aa753 in 4-2.J41 and aa759 in JRCSF, which includes a conserved hydrophilic domain (CHD), restores native-like conformation of these Envs on the plasma membrane. However, CT-deletion in 4-2.J41 and JRCSF at the pseudovirus level has either no or only modest effect on neutralization potency. CONCLUSION: Here, we report that the CHD in the CT of Env plays an important role in regulating the ET integrity of a subset of efficiently cleaved, functional Envs on the cell surface.

Characterization of the membrane-bound form of the chimeric, B/C recombinant HIV-1 Env, LT5.J4b12C.

Human immunodeficiency virus 1 (HIV-1) diversity is a significant challenge in developing a vaccine against the virus. B/C recombinants have been found in India and other places but are the predominant clade prevalent in China. HIV-1 envelopes (Envs) are the target of broadly neutralizing antibodies (bNAbs) which develop spontaneously in some HIV-1 infected patients. It has been previously reported with efficiently cleaved clade A, B and C Envs that preferential binding of Envs to bNAbs as opposed to non-NAbs, a desirable property for immunogens, is correlated with efficient cleavage of the Env precursor polypeptide into constituent subunits. These Envs are suitable for designing immunogens as soluble proteins, virus-like particles or for delivery by viral vectors/plasmid DNA. However, a B/C recombinant Env with similar properties has not been reported. Here we show that the chimeric, recombinant B/C clade Env LT5.J4b12C is efficiently cleaved on the plasma membrane and selectively binds to bNAbs.

Membrane bound modified form of clade B Env, JRCSF is suitable for immunogen design as it is efficiently cleaved and displays all the broadly neutralizing epitopes including V2 and C2 domain-dependent conformational epitopes.

BACKGROUND: Antigenicity of HIV-1 envelope proteins (Envs) of both lab-adapted and primary isolates expressed on the cell surface rarely match with in vitro neutralization of viruses, pseudo-typed with corresponding Envs. Often, both neutralizing and non-neutralizing antibodies bind to Envs expressed on the cell membrane. This could be due to the lack of efficient cleavage of Env expressed on the cell surface. Naturally occurring, efficiently cleaved Envs with appropriate antigenic properties are relatively rare. Given viral diversity it is essential to increase the pool of candidate Envs suitable for immunogen design. Previously, it has been reported that JRFL Env is the only clade B Env, which is efficiently cleaved on the cell surface and retains desirable antigenic properties. JRCSF is a clade B Env isolated from the same patient as JRFL. JRCSF Env has not been explored aggressively for designing immunogen as the binding characteristics of JRCSF Env to broadly neutralizing antibodies on the cell surface and its cleavage status are unknown. RESULTS: Although JRCSF preferentially binds to most of the other gp120-directed neutralizing antibodies and cleavage dependent antibody, PGT151 efficiently, it binds poorly to CD4-binding-site-directed (CD4-bs-directed) neutralizing antibodies on cell surface. Membrane bound form of modified JRCSF Env containing the N197D mutation binds to CD4-bs-directed neutralizing antibodies better than JRFL, without debilitating its ability to bind quaternary epitope-directed neutralizing antibodies or exposing the CD4i antibody epitopes. In comparison to JRFL (E168K), JRCSF Env binds more efficiently to PG9/PGT145 class of V1/V2-directed conformational antibodies. Biochemical, cell surface staining and gp120 shedding experiments suggest that JRCSF is efficiently cleaved on the cell surface. CONCLUSIONS: Binding of JRCSF Env expressed on cell surface to the various HIV-1 Env-directed antibodies has not been reported earlier. Here, for the first time, we report that compared to JRFL, JRCSF displays epitopes for a larger number of broadly neutralizing antibodies and is also efficiently cleaved when expressed on the cell surface. Thus, considering the diversity of viral Envs and the discovery of conformation dependent glycan-directed antibodies in HIV-1 infected individuals, an innately cleaved JRCSF Env as present on the viral membrane and displaying those distinct epitopes may be an important candidate for immunogen design.

Structural insights into trypanosomatid Mnk kinase orthologues (kMnks) suggest altered mechanism in the kinase domain.

Mitogen-activated protein kinase (MAPK) interacting protein kinases (Mnk1 and Mnk2) mediated phosphorylation of the eukaryotic initiation factor eIF4E is an important translation initiation control, in Mnk-mediated oncogenic activity and other disease conditions. Thus, Mnk kinases are an important target for therapy. Trypanosomatids are a class of kinetoplastids, some of which are protozoan parasites and cause diseases in humans. While protein translation initiation is well understood in eukaryotes and prokaryotes, there is a lack of sufficient structural information of this process in trypanosomatids. Here, we report that trypanosomatids have one orthologue of Mnk kinase with low overall sequence homology but high homology in the kinase domain and an additional C-terminal domain containing putative calmodulin binding site(s). We show that while many of the domains and motifs are conserved, homology modeling/structure prediction, docking analysis and molecular dynamics simulation studies suggest that trypanosomatid kMnk kinases, kinase domains are present in DFG-in conformation as opposed to the auto-inhibited DFD-out conformation of un-phosphorylated human Mnk1. Furthermore, we observed that several regulatory features are different in trypanosomatid kMnk kinases. Our study indicates that mechanism and regulation in the kinase domain of trypanosomatid kMnks are likely to be altered, and that they can be important drug targets.

Architecture, domain organization, and functional signatures of trypanosomatid keIF4A1s and Plasmodium peIF4A1s suggest conserved functions.

Initiation of translation is the first of the three obligatory steps required for protein synthesis and is carried out by a large number of protein factors called initiation factors in conjunction with ribosomes. One of the key conserved protein factors in eukaryotes that plays a role in this process is eIF4A, which has three homologues in humans with eIF4A1 being the primary factor playing a role in translation initiation. eIF4As are members of the family of DEAD-box helicases that carry out different biological functions. eIF4A1s are recruited to translation initiation complexes via association with eIF4G and have ATP binding, ATP hydrolysis, RNA binding, and unwinding activities. Plasmodium and trypanosomatids such as Leishmania and Trypanosoma are parasites that cause human disease. While mechanistically the function of eIF4A1s in eukaryotes is wellunderstood, the orthologues peIF4A1s and keIF4A1s in Plasmodium and trypanosomatids are not well-studied. Here, we have used bioinformatics tools and homology modelling/structure prediction to study the motifs and functional signatures of Plasmodium and trypanosomatid peIF4A1s/keIF4A1s. We report a high degree of sequence conservation, structural conservation, and conservation of protein-protein interaction signatures of Plasmodium and trypanosomatid peIF4A1s/keIF4A1s in comparison with human eIF4A1. Thus, in spite of the great divergence in evolution between these parasites and higher eukaryotes, there is remarkable conservation of motifs and functional signatures in Plasmodium and trypanosomatid peIF4A1s/keIF4A1s.

Kennedy Epitope (KE)-dependent Retrograde Transport of Efficiently Cleaved HIV-1 Envelopes (Envs) and its Effect on Env Cell Surface Expression and Viral Particle Formation.

Efficiently cleaved HIV-1 Envs are the closest mimics of functional Envs as they specifically expose only bNAb (broadly neutralizing antibody) epitopes and not non-neutralizing ones, making them suitable for developing vaccine immunogens. We have previously identified several efficiently cleaved Envs from clades A, B, C and B/C. We also described that truncation of the CT (C-terminal tail) of a subset of these Envs, but not others, impairs their ectodomain conformation/antigenicity on the cell surface in a CT conserved hydrophilic domain (CHD) or Kennedy epitope (KE)-dependent manner. Here, we report that those Envs (4 - 2.J41 and JRCSF), whose native-like ectodomain conformation/antigenicity on the cell surface is disrupted upon CT truncation, but not other Envs like JRFL, whose CT truncation does not have an effect on ectodomain integrity on the cell surface, are also defective in retrograde transport from early to late endosomes. Restoration of the CHD/KE in the CT of these Envs restores wild-type levels of distribution between early and late endosomes. In the presence of retrograde transport inhibitor Retro 2, cell surface expression of 4 - 2.J41 and JRCSF Envs increases [as does in the presence of Rab7a DN and Rab7b DN (DN: dominant negative)] but particle formation decreases for 4 - 2.J41 and JRCSF Env pseudotyped viruses. Our results show for the first time a correlation between CT-dependent, CHD/KE regulated retrograde transport and cell surface expression/viral particle formation of these efficiently cleaved Envs. Based on our results we hypothesize that a subset of these efficiently cleaved Envs use a CT-dependent, CHD/KE-mediated mechanism for assembly and release from late endosomes.

Opposing action of casein kinase 1 and calcineurin in nucleo-cytoplasmic shuttling of mammalian translation initiation factor eIF6.

Eukaryotic initiation factor 6 (eIF6), a highly conserved protein from yeast to mammals, is essential for 60 S ribosome biogenesis and assembly. Both yeast and mammalian eIF6 are phosphorylated at Ser-174 and Ser-175 by the nuclear isoform of casein kinase 1 (CK1). The molecular basis of eIF6 phosphorylation, however, remains elusive. In the present work, we show that subcellular distribution of eIF6 in the nuclei and the cytoplasm of mammalian cells is mediated by dephosphorylation and phosphorylation, respectively. This nucleo-cytoplasmic shuttling is dependent on the phosphorylation status at Ser-174 and Ser-175 of eIF6. We demonstrate that Ca(2+)-activated calcineurin phosphatase binds to and promotes nuclear localization of eIF6. Increase in intracellular concentration of Ca(2+) leads to rapid translocation of eIF6 from the cytoplasm to the nucleus, an event that is blocked by specific calcineurin inhibitors cyclosporin A or FK520. Nuclear export of eIF6 is regulated by phosphorylation at Ser-174 and Ser-175 by the nuclear isoform of CK1. Mutation of eIF6 at the phosphorylatable Ser-174 and Ser-175 to alanine or treatment of cells with the CK1 inhibitor, D4476 inhibits nuclear export of eIF6 and results in nuclear accumulation of eIF6. Together, these results establish eIF6 as a substrate for calcineurin and suggest a novel paradigm for calcineurin function in 60 S ribosome biogenesis via regulating the nuclear accumulation of eIF6.

Generation of soluble, cleaved, well-ordered, native-like dimers of dengue virus 4 envelope protein ectodomain (sE) suitable for vaccine immunogen design.

Dengue virus is transmitted by Aedes mosquitoes and dengue is endemic in many regions of the world. Severe dengue results in complications that may lead to death. Although some vaccine candidates are in clinical trials and one vaccine Dengvaxia, with restricted efficacy, is available, there are currently no specific therapies to completely prevent or treat dengue. The dengue virus structural protein E (envelope) exists as a head-to-tail dimer on mature virus, is targeted by broadly neutralizing antibodies and is suitable for developing vaccine immunogens. Here, we have used a redesigned dengue prME expression construct and immunoaffinity chromatography with conformational/quaternary antibody A11 to purify soluble DENV4 sE(A259C) (E ectodomain) dimers from mammalian expression system to ~99 % purity. These dimers retain glycosylation reported for native DENV E, display the three major broadly neutralizing antibody epitopes, and form well-ordered structure. This strategy can be used for developing subunit vaccine candidates against dengue and other flaviviruses.

Recruitment of a SAP18-HDAC1 complex into HIV-1 virions and its requirement for viral replication.

HIV-1 integrase (IN) is a virally encoded protein required for integration of viral cDNA into host chromosomes. INI1/hSNF5 is a component of the SWI/SNF complex that interacts with HIV-1 IN, is selectively incorporated into HIV-1 (but not other retroviral) virions, and modulates multiple steps, including particle production and infectivity. To gain further insight into the role of INI1 in HIV-1 replication, we screened for INI1-interacting proteins using the yeast two-hybrid system. We found that SAP18 (Sin3a associated protein 18 kD), a component of the Sin3a-HDAC1 complex, directly binds to INI1 in yeast, in vitro and in vivo. Interestingly, we found that IN also binds to SAP18 in vitro and in vivo. SAP18 and components of a Sin3A-HDAC1 complex were specifically incorporated into HIV-1 (but not SIV and HTLV-1) virions in an HIV-1 IN-dependent manner. Using a fluorescence-based assay, we found that HIV-1 (but not SIV) virion preparations harbour significant deacetylase activity, indicating the specific recruitment of catalytically active HDAC into the virions. To determine the requirement of virion-associated HDAC1 to HIV-1 replication, an inactive, transdominant negative mutant of HDAC1 (HDAC1(H141A)) was utilized. Incorporation of HDAC1(H141A) decreased the virion-associated histone deacetylase activity. Furthermore, incorporation of HDAC1(H141A) decreased the infectivity of HIV-1 (but not SIV) virions. The block in infectivity due to virion-associated HDAC1(H141A) occurred specifically at the early reverse transcription stage, while entry of the virions was unaffected. RNA-interference mediated knock-down of HDAC1 in producer cells resulted in decreased virion-associated HDAC1 activity and a reduction in infectivity of these virions. These studies indicate that HIV-1 IN and INI1/hSNF5 bind SAP18 and selectively recruit components of Sin3a-HDAC1 complex into HIV-1 virions. Furthermore, HIV-1 virion-associated HDAC1 is required for efficient early post-entry events, indicating a novel role for HDAC1 during HIV-1 replication.

Chikungunya and arthritis: An overview.

Chikungunya is caused by CHIKV (chikungunya virus), an emerging and re-emerging arthropod-vectored viral infection that causes a febrile disease with primarily long term sequelae of arthralgia and myalgia and is fatal in a small fraction of infected patients. Sporadic outbreaks have been reported from different parts of the world chiefly Africa, Asia, the Indian and Pacific ocean regions, Europe and lately even in the Americas. Currently, treatment is primarily symptomatic as no vaccine, antibody-mediated immunotherapy or antivirals are available. Chikungunya belongs to a family of arthritogenic alphaviruses which have many pathophysiological similarities. Chikungunya arthritis has similarities and differences with rheumatoid arthritis. Although research into arthritis caused by these alphaviruses have been ongoing for decades and significant progress has been made, the mechanisms underlying viral infection and arthritis are not well understood. In this review, we give a background to chikungunya and the causative virus, outline the history of alphavirus arthritis research and then give an overview of findings on arthritis caused by CHIKV. We also discuss treatment options and the research done so far on various therapeutic intervention strategies.

Design and characterization of a germ-line targeting soluble, native-like, trimeric HIV-1 Env lacking key glycans from the V1V2-loop.

BACKGROUND: The HIV-1 envelope glycoprotein (Env) is the primary target for broadly neutralizing antibodies (bNAbs) which can block infection. The current design strategy of soluble forms of Env in native-like trimeric conformation induces neutralizing antibodies with minimal breadth and potency. Extensive shielding by N-glycans on the surface of the HIV-1 Env acts as an immune evasion mechanism by restricting B cell recognition of conserved neutralizing determinants. An alternate approach is to design Env protein with glycan deletion to expose the protein surface. METHODS: A stable native-like trimeric Env with glycan holes at potentially immunogenic locations is expected to elicit better induction of germ-line B-cells due to exposure of the immunogenic regions. However, the extent and consequences of glycan removal from the trimer apex that form an important epitope is not explored. In this work, we have designed a construct with glycans deleted from the trimer apex of an Indian clade C origin Env that has previously been characterized for immunogenicity, to understand the impact of deglycosylation on the structural and functional integrity as well as on the antibody binding properties. RESULTS: The V1V2 glycan-deleted protein maintains native-like trimeric conformation with improved accessibility of the V1V2-directed germ-line antibodies. Furthermore, we showed that the protein binds specifically to quaternary conformation-dependent bnAbs but minimally to non-neutralizing antibodies. CONCLUSIONS: This study provide an important design aspect of HIV-1 Env-based immunogens with glycan holes in the apex region that could be useful in eliciting apex directed antibodies in immunization studies.

The SARS CoV-2 spike directed non-neutralizing polyclonal antibodies cross-react with Human immunodeficiency virus (HIV-1) gp41.

Cross-reactivity among the two diverse viruses is believed to originate from the concept of antibodies recognizing similar epitopes on the two viral surfaces. Cross-reactive antibody responses have been seen in previous variants of SARS and SARS-CoV-2, but little is known about the cross reactivity with other similar RNA viruses like HIV-1. In the present study, we examined the reactivity the SARS-CoV-2 directed antibodies, via spike, immunized mice sera and demonstrated whether they conferred any cross-reactive neutralization against HIV-1. Our findings show that SARS-CoV-2 spike immunized mice antibodies cross-react with the HIV-1 Env protein. Cross-neutralization among the two viruses is uncommon, suggesting the presence of a non-neutralizing antibody response to conserved epitopes amongst the two viruses. Our results indicate, that SARS-CoV-2 spike antibody cross reactivity is targeted towards the gp41 region of the HIV-1 Env (gp160) protein. Overall, our investigation not only answers a crucial question about the understanding of cross-reactive epitopes of antibodies generated in different viral infections, but also provides critical evidence for developing vaccine immunogens and novel treatment strategies with enhanced efficacy capable of recognising diverse pathogens with similar antigenic features.

Non-neutralizing SARS CoV-2 directed polyclonal antibodies demonstrate cross-reactivity with the HA glycans of influenza virus.

The spike protein of the SARS-CoV-2 virus is the foremost target for the designing of vaccines and therapeutic antibodies and also acts as a crucial antigen in the assessment of COVID-19 immune responses. The enveloped viruses; such as SARS-CoV-2, Human Immunodeficiency Virus-1 (HIV-1) and influenza, often hijack host-cell glycosylation pathways and influence pathobiology and immune selection. These glycan motifs can lead to either immune evasion or viral neutralization by the production of cross-reactive antibodies that can lead to antibody-dependent enhancement (ADE) of infection. Potential cross-protection from influenza vaccine has also been reported in COVID-19 infected individuals in several epidemiological studies recently; however, the scientific basis for these observations remains elusive. Herein, we show that the anti-SARS-CoV2 antibodies cross-reacts with the Hemagglutinin (HA) protein. This phenomenon is common to both the sera from convalescent SARS-CoV-2 donors and spike immunized mice, although these antibodies were unable to cross-neutralize, suggesting the presence of a non-neutralizing antibody response. Epitope mapping suggests that the cross-reactive antibodies are targeted towards glycan epitopes of the SARS-CoV-2 spike and HA. Overall, our findings address the cross-reactive responses, although non-neutralizing, elicited against RNA viruses and warrant further studies to investigate whether such non-neutralizing antibody responses can contribute to effector functions such as antibody-dependent cellular cytotoxicity (ADCC) or ADE.